WO2021217251A1 - Multimères de protéines auto-assemblés immobilisés - Google Patents

Multimères de protéines auto-assemblés immobilisés Download PDF

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Publication number
WO2021217251A1
WO2021217251A1 PCT/CA2021/050573 CA2021050573W WO2021217251A1 WO 2021217251 A1 WO2021217251 A1 WO 2021217251A1 CA 2021050573 W CA2021050573 W CA 2021050573W WO 2021217251 A1 WO2021217251 A1 WO 2021217251A1
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WIPO (PCT)
Prior art keywords
amino acid
chimeric polypeptide
polypeptide
aat
bat
Prior art date
Application number
PCT/CA2021/050573
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English (en)
Inventor
Heyu Ni
Miguel Neves
Original Assignee
CCOA Therapeutics Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by CCOA Therapeutics Inc. filed Critical CCOA Therapeutics Inc.
Priority to CA3181186A priority Critical patent/CA3181186A1/fr
Priority to US17/997,090 priority patent/US20230331811A1/en
Priority to CN202180031433.1A priority patent/CN115956090A/zh
Publication of WO2021217251A1 publication Critical patent/WO2021217251A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70546—Integrin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/14—Peptides being immobilised on, or in, an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • the present disclosure relates to surfaces having a polypeptide multimer, such as a polypeptide heterodimer, immobilized thereon and maintaining the biological activity of the heterodimer.
  • Immobilized proteins are important tools in proteomics and diagnosis, allowing one to obtaining information about protein functions and interactions. Immobilized proteins and enzymes are very important for commercial uses as they possess many benefits to the expenses and processes of the associated reaction, including convenience of use, reusability, and greater stability compares to soluble forms of the proteins and enzymes. Ideally, proteins need to be immobilized onto surfaces while maintaining their biological activity, optionally with high density in order to allow the usage of small amount of sample solution. Nonspecific protein adsorption also need to be avoided or at least minimized in order to improve detection performances.
  • the present disclosure provides biologically active forms of multimers (such as heterodimers) on a surface using charged tails.
  • the charged tails immobilizes the multimers onto the surface, while still allowing the monomeric protein units of the multimers to interact with each other.
  • the charged tails also interact with one another.
  • a surface having a first and a second hydroxyl group and at least one self-assembled multimer immobilized thereon, wherein: the at least one self-assembled multimer comprises at least one a first chimeric polypeptide associated with a second chimeric polypeptide.
  • the first chimeric polypeptide is of formula (la) or (lb)
  • the second chimeric polypeptide is of formula (lla) or (lib):
  • the second chimeric polypeptide is of formula (lla) or (lib) :
  • SPM is a second polypeptide moiety
  • SAAL is an optional second amino acid linker
  • BAT is a basic amino acid tail having at least one acid amino acid residue having an R-group comprising a carboxyl group, and wherein the BAT has a pi between about 9 and 11
  • - is an amine bond.
  • the carboxyl group of the first chimeric polypeptide is covalently associated to a first silane linker (FSL) moiety, wherein the FSL is covalently associated with a first hydroxyl group of the surface.
  • FSL silane linker
  • the carboxyl group of the second chimeric polypeptide is covalently associated to a second silane linker (SSL) moiety, wherein the SSL is covalently associated with a second hydroxyl group of the surface.
  • the AAT is non-covalently associated with the BAT.
  • the first chimeric polypeptide is non-covalently associated with the second polypeptide moiety.
  • the FPM and the SPM are the same and the multimer is a homodimer, a homotrimer or a homomultimer comprising more than three identical polypeptide moieties.
  • the FPM and the SPM are different and the multimer is an heterodimer, a heterotrimer or a heteromultimer comprising additional polypeptide moieties.
  • the AAT is at least three and up to 50 amino acid residues in length. In a further embodiment, wherein the AAT has a pi is about 4. In yet another embodiment, wherein the AAT has a pi of about 3.91. In some embodiments, the AAT has an amino acid sequence of SEQ ID NO: 4 or functional variants or fragments thereof. In an embodiment, the BAT is at least one and up to 50 amino acid residues in length. In a further embodiment, the BAT has a pi is about. In yet a further embodiment, the BAT has a pi of about 10.1. In still another embodiment, the BAT has an amino acid sequence of SEQ ID NO: 9 or functional variants or fragments thereof.
  • the first chimeric polypeptide has the FAAL and/or the second chimeric polypeptide has the SAAL.
  • the at least one self-assembled multimer comprising an ectodomain of a surface protein.
  • one or more of the at least one self-assembled multimer is an activated surface protein.
  • the at least one self-assembled multimer is an integrin dimer.
  • the first chimeric polypeptide comprises a allb polypeptide
  • the second chimeric polypeptide comprises a b3 polypeptide.
  • the FPM has an amino acid sequence of SEQ ID NO: 2 or functional variants or fragments thereof and/or the SPM has an amino acid sequence of SEQ ID NO: 7 or functional variants or fragments thereof.
  • the surface is a spherical surface, such as, for example, a microsphere (e.g., a microsphere silica bead).
  • the surface comprises a planar surface.
  • the FSL and/or SSL comprise one or more amine or thiol groups that are covalently associated with the carboxyl groups of the AAT or the BAT.
  • the FSL and/or SSL moieties comprise (3- trimethoxysilylpropyl) diethylenetriamine (DETA).
  • the surface comprises the first chimeric polypeptide of formula (la) and the second chimeric polypeptide of formula (lla); the first chimeric polypeptide of formula (lb) and the second chimeric polypeptide of formula (lla); the first chimeric polypeptide of formula (la) and the second chimeric polypeptide of formula (lib); or the first chimeric polypeptide of formula (lb) and the second chimeric polypeptide of formula (lib).
  • a process of immobilizing at least one self-assembled multimer to a surface having a first and a second hydroxyl groups covalently associated with a first and a second silane linker moiety comprises a first and a second chimeric polypeptide.
  • the process comprises obtaining the first chimeric polypeptide as described herein; obtaining the second chimeric polypeptide as described herein; and adding the first and the second chimeric polypeptide to the surface in a solvent under suitable conditions for the first and second chimeric polypeptides to covalently bond to the surface via the silane linker moieties.
  • the AAT of the first chimeric polypeptide is non-covalently associated with the BAT of the second chimeric polypeptide and the first polypeptide moiety is non-covalently associated with the second polypeptide moiety.
  • first and second silane linker moieties comprise one or more amine or thiol groups that are covalently associated with the carboxyl groups of the AAT or BAT.
  • first and/or second silane linker moieties comprise (3-trimethoxysilylpropyl) diethylenetriamine (DETA).
  • the process further comprises coating the surface with the silane linker moieties by reacting with the hydroxyl groups.
  • the process further comprises obtaining the first and the second chimeric polypeptide from recombinant expression in a recombinant host cell.
  • the process further comprises activating the at least one self-assembled multimer.
  • the process comprises incubating the surface having the first and second chimeric polypeptides bonded thereon in an activation buffer comprising cations.
  • the activation buffer comprises divalent cations.
  • the at least one self- assembled multimer comprises an ectodomain of a surface protein.
  • the at least one self-assembled multimer is an integrin dimer.
  • the surface is a microsphere or is flat.
  • kits comprising (i) a first and (ii) a second chimeric polypeptide as described herein, wherein the first and the second chimeric polypeptide are capable of forming a multimer and optionally (iii) a surface for covalently associating the first and the second chimeric polypeptide, wherein the surface has hydroxyl groups covalently associated with a first and a second silane linker moiety.
  • the first chimeric polypeptide has a first polypeptide moiety (FPM), an optional first amino acid linker (FAAL), and a first amino acid tail (AAT) having at least three acid amino acid residue having an R-group comprising a carboxyl group, and wherein the AAT has a pi between about 3 and 5 and the second chimeric polypeptide has a second polypeptide moiety (SPM), an optional second amino acid linker (SAAL), and a second amino acid tail (BAT) having at least one acid amino acid residue having an R-group comprising a carboxyl group, and wherein the BAT has a pi between about 9 and 11.
  • the FPM is a allb polypeptide and the SPM is a b3 polypeptide.
  • the first chimeric polypeptide can have an amino acid sequence of SEQ ID NO: 1 or functional variants or fragments thereof and/or the second chimeric polypeptide can have an amino acid sequence of SEQ ID NO: 6 or functional variants or fragments thereof.
  • the surface is a microsphere, such as, for example, a microsphere silica bead (which can be coated with the silane linker moiety). In still another embodiment, the surface is flat (which can be coated with the silane linker moiety).
  • the silane linker moieties comprise one or more amine or thiol groups that are covalently associated with the carboxyl groups of the AAT or BAT.
  • the silane linker moiety is (3- trimethoxysilylpropyl) diethylenetriamine (DETA).
  • FIG. 1A Generalized representation of unimolecular self-assembled monolayer (SAM) surface assembly. Linkers consisting of a backbone, flanked by a tail and a head group, assemble with the tails attached to the silica and the heads available for covalent probe attachment.
  • FIG. 1B The different conformation states of an integrin are presented. In the inactive (“Bent”) and intermediate (“Extended”) conformations, integrins have no/low affinity towards their target. In the high-affinity ligand binding (“Open”) conformation, integrins have affinity towards their target. For each pair of heterodimer shown, left heterodimer is the a-subunit and the right heterodimer is the b-subunit.
  • FIG. 1C Illustration of the formation of a (3-trimethoxysilylpropyl) diethylenetriamine (DETA) SAM onto a cleaned silica substrate (step 1), coupling of recombinant human ectodomain ⁇ ll ⁇ 3 onto DETA-SAM (step 2) which involves site-specific coupling of recombinant human ectodomain ⁇ ll ⁇ 3 having acidic and basic tails onto DETA SAMs (step 2a), and hydrolysis of any unreacted NHS groups to stop the surface coupling reaction by immersing the substrate in borate buffer pH 8.5 (step 2b). Activation of immobilized recombinant human ectodomain ⁇ ll ⁇ 3 into high-affinity ligand binding conformation (step 3). For each pair of heterodimer shown, left heterodimer is allb and the right heterodimer is b3.
  • DETA 3-trimethoxysilylpropyl) diethylenetriamine
  • Figures 2A to 2I show the characterization of ⁇ ll ⁇ 3-coupled silica particles by comparing bare, DETA coated, inactive ⁇ ll ⁇ 3 coupled and activated ⁇ ll ⁇ 3 coupled beads with fluorescently coupled antibodies and ligands.
  • FIG. 2A flow cytometry histograms showing PSI-E1 binding (conformation independent ⁇ ll ⁇ 3 mAb)
  • FIG. 2B corresponding mean fluorescence intensities (MFI) from Fig. 2A plotted in bar graphs
  • Fig. 2C flow cytometry histograms showing fibrinogen (endogenous ligand of aI ⁇ b3) binding
  • Fig. 2D corresponding mean fluorescence intensities (MFI) from Fig.
  • FIG. 2C plotted in bar graphs
  • FIG. 2E fibrinogen dose-response curves
  • FIG. 2F flow cytometry dot plots and corresponding MFI bar graphs of DETA and activated ⁇ ll ⁇ 3 particles binding fibrinogen before and after treatment with 1 mM EDTA
  • FIG. 2G flow cytometry dot plots of (left to right) DETA, Preactivation and activated ⁇ ll ⁇ 3 particles analyzed for PAC-1 binding
  • FIG. 2H corresponding mean fluorescence intensities (MFI) from Fig. 2G plotted in bar graphs
  • FIG. 2H mean fluorescence intensities
  • Figures 3A and 3B show ⁇ ll ⁇ 3 is covalently bound to the surface of the beads and covalent binding increases the anti-fouling and fibrinogen binding properties of the aI ⁇ b3- coupled beads.
  • Fig. 3A DETA coated, ⁇ ll ⁇ 3 coupled beads with FITC-coupled PSI-E1 (conformation independent ⁇ ll ⁇ 3 mAb) in the absence (left panel) and presence (right panel) of SDS.
  • Fig. 3B DETA coated, ⁇ ll ⁇ 3 coupled beads with fibrinogen (endogenous ligand of ⁇ ll ⁇ 3). Results are provided as the mean fluorescence intensity on the Y-axis, and adsorbed or covalent ⁇ ll ⁇ 3 (as well as active or inactive conformation) on the X-axis.
  • Figures 4A and 4B show co-aggregation of ⁇ ll ⁇ 3-coupled beads with (Fig. 4A) wild type and (Fig. 4B) Fibrinogen/von Willebrand factor double deficient (Fg/VWF gel-filtered platelets.
  • White arrows indicates platelets and black arrows indicates ⁇ ll ⁇ 3 coated beads. Scale bar 20 pm.
  • Figures 5A and 5B compares (Fig. 5A) quantitative flow cytometry fibrinogen assay versus (Fig. 5B) fibrinogen ELISA. n ⁇ 3.
  • Fig. 5A X-axis is fibrinogen concentration in ⁇ M
  • Y-axis is mean fluorescence intensity
  • Fig. 5B X-axis is fibrinogen concentration in ⁇ M
  • Y-axis is absorbance at 492 nm.
  • Figure 6 shows the optimization of the loading of recombinant human ⁇ ll ⁇ 3 ectodomain onto the DETA coated silica surface by evaluating the amount of integrin loaded on the surface against the binding activity against its cognate ligand, fibrinogen. Results are shown as flow cytometry signal associate with fibrinogen binding (left axis, grey line and ⁇ ) or with PSI-E1 binding (right axis, black line and ⁇ ) in function of the concentration of ⁇ ll ⁇ 3 ectodomain immobilized onto the DETA coated silica surface.
  • FIG. 7A Platelet aggregates form under the conditions used for the platelet-bead co-aggregation experiments. Left panel shows the bright field color. Right panel shows the bright field.
  • Fig. 7B DETA beads in the presence of fibrinogen, and ⁇ ll ⁇ 3 coated beads without fibrinogen do not aggregate. While ⁇ ll ⁇ 3 coated beads in the presence of fibrinogen aggregate. Left set of panels shows the bright field color. Right set of panels shows bright field with fluorescence overlay to clearly show aggregates.
  • the present disclosure relates to multimeric polypeptides, such as homo- or heteromultimers, such as homo- or heterodimers (which can include, in some embodiments, one or more ectodomains), immobilized on surfaces that maintain their function and active conformation.
  • the polypeptides are provided in the form of chimeric polypeptides having an amino acid tail for covalently associating with the surface, thereby immobilizing the chimeric polypeptides to the surface so as to form the multimer.
  • Figure 1A provides a representation of unimolecular self-assembly entities which can be combined to form the mutlimeric polypeptides.
  • multimeric polypeptides can be provided in an inactive or “bent” conformation, an intermediate or “extended” conformation and, upon the binding of the cognate ligand, a high affinity or “open” conformation ( Figure 1B).
  • the multimeric polypeptides of the present disclosure seek to be provided in an activable or an active confirmation.
  • the amino acid tail has at least one acid amino acid residue having an R-group comprising a carboxyl group.
  • Unnatural amino acids having a side chain R-group include, for example, amino acids with dextrorotary (D)-configuration or amino acids with synthetic or variant R- groups (termed non-natural amino acids) that have been modified to add a terminal or nonterminal carboxyl group.
  • D dextrorotary
  • R- groups synthetic or variant R- groups
  • the person skilled in the art will recognize that the amino acid residues present on the tail of each of the chimeric polypeptide is not limited to a particular naturally-occurring or synthetic amino acid residues.
  • the amino acid tail is attached to the polypeptide (either directly or indirectly using a linker) in such a way that the polypeptide maintains its conformation, functionality or biological activity.
  • the amino acid tail is attached (either directly or indirectly using a linker) to one end (carboxyl- or amino-end) of the polypeptide.
  • the amino acid tail is attached to an end of the polypeptide that is opposite to the functional end of the polypeptide to avoid loss of conformation, functionality or biological activity of the polypeptide. For example, if the polypeptide bears its biological activity at the carboxyl-end, the amino acid tail is going to be attached to the amino-end of the polypeptide.
  • the amino acid tail is going to be attached to the carboxyl-end of the polypeptide.
  • the amino acid tail is attached to the carboxyl end of the polypeptide. In other embodiments, the amino acid tail is attached to the amino end of the polypeptide.
  • one of the carboxyl group of the polypeptide (which can be associated with the amino acid tail) is covalently associated with a silane linker.
  • the carboxyl group of the R-group of one of the amino acid residue of an amino acid tail is for covalent association (a chemical bond) with a silane linker moiety which is immobilized on the surface.
  • the silane linker moiety has one or more terminal or non-terminal the amine (-NH 2 -) or thiol (-S-) groups for covalent association or chemical bonding with the carboxyl group of the polypeptide and, in some embodiments, of the acidic amino acid residue(s) of the amino acid tail associated to the polypeptide.
  • polypeptides of the present disclosure can be presented as chimeric polypeptides.
  • the chimeric polypeptides can have formula (Ilia) or (lllb):
  • the chimeric polypeptides can have formula (IVa) or (IVb):
  • amino acid linker AAL
  • amino acid tail is directly associated with the polypeptide moiety.
  • carboxyl terminus of the polypeptide moiety is directly associated (with an amide linkage) to the amino terminus of the amino acid tail.
  • carboxyl terminus of the amino acid tail is directly associated (with an amide linkage) to the amino terminus of the polypeptide moiety.
  • an amino acid linker is desirable either to provide, for example, some flexibility between the polypeptide moiety and the amino acid tail or to facilitate the construction of the chimeric polypeptide (which can, in some embodiments, be encoded by a nucleic acid molecule).
  • the “amino acid linker” or “AAL” refer to a stretch of one or more amino acids separating the polypeptide moiety (PM) and the amino acid tail (AT) (e.g., indirectly linking the polypeptide moiety to the amino acid tail). It is preferred that the amino acid linker be neutral, e.g., does not interfere with the biological activity of the polypeptide moiety nor with the biological or chemical activity or interactions of the amino acid tail.
  • amino acid linker (AAL) In instances in which the amino acid linker (AAL) is present in the chimeras of formula (Ilia or IVa), its amino end is associated (with an amide linkage) to the carboxyl end of the polypeptide moiety and its carboxyl end is associated (with an amide linkage) to the amino end of the amino acid tail. In instances in which the amino acid linker (AAL) is present in the chimeras of formula (lllb of IVb), its amino end is associated (with an amide linkage) to the carboxyl end of the amino acid tail and its carboxyl end is associated (with an amide linkage) to the amino end of the polypeptide moiety.
  • the amino acid linker is (GGGS) n (also referred to as G 3 S) and in still further embodiments, the amino acid linker L comprises more than one G 3 S motifs.
  • the amino acid linker can be (G 3 S) 3 and have the amino acid sequence of SEQ ID NO: 3.
  • the amino acid tail (AT) is at least one amino acid and up to 50 amino acid residues in length. In some embodiments, the amino acid tail (AT) is at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid residues long.
  • the amino acid tail is no more than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41 , 40, 39, 38, 37, 36, 35, 34, 33, 32, 31 , 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues long.
  • chimeric multimer polypeptides are provided to immobilize multimers on surfaces while maintaining their function and, in some embodiments, active conformation with respect to each other.
  • a surface is provided having hydroxyl groups and at least one self-assembled multimer immobilized thereon.
  • the multimer comprises at least one first chimeric polypeptide associated with a second chimeric polypeptide.
  • the multimer can be a homo-multimer or a hetero-multimer.
  • the first chimeric polypeptide is of formula (la) or (lb) :
  • FPM is a first polypeptide moiety
  • FAAL is an optional first amino acid linker
  • AAT is an acidic amino acid tail having at least three acidic amino acid residues having an R-group comprising a carboxyl group.
  • the AAT has a pi between about 3 and 5; and - is an amine bond.
  • the first chimeric polypeptide is of formula (Va) or (Vb):
  • AAT is an acidic amino acid tail having at least three acidic amino acid residues having an R-group comprising a carboxyl group.
  • the AAT has a pi between about 3 and 5.
  • the - is an amine bond.
  • the FPM includes an ectodomain (and in some additional embodiments, a complete ectodomain) of a surface polypeptide.
  • an ectodomain is the domain of a membrane protein that extends in the extracellular space.
  • the ectodomain is involved in binding a ligand and can lead to signal transduction.
  • an “acidic amino acid tail” refers to an amino acid tail having one or more acidic amino acid residues, such that the pi of the acidic amino acid tail is less than 7.
  • acidic amino acid residues include: aspartic acid and glutamic acid.
  • the pi of the acidic amino acid tail is less between about 3 and 5.
  • the pi of the acidic amino acid tail is about 4, more preferably 3.91.
  • the acidic amino acid tail has a charge which will allow it to interact non-covalently with the basic amino acid tail (described below) and place the FPM is an active conformation.
  • the acidic amino acid tail (AAT) is at least three amino acid and up to 50 amino acid residues in length. In some embodiments, the acidic amino acid tail (AAT) is 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid residues long.
  • the acidic amino acid tail is no more than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41 , 40, 39, 38, 37, 36, 35, 34, 33, 32, 31 , 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4 or 3 amino acid residues long.
  • the AAT has an amino acid sequence of SEQ ID NO: 4 or variants or fragments thereof.
  • the first amino acid linker (FAAL) is present in the chimeras of formula (la)
  • its amino end is associated (with an amide linkage) to the carboxyl end of the first polypeptide moiety and its carboxyl end is associated (with an amide linkage) to the amino end of the acidic amino acid tail.
  • the first amino acid linker (FAAL) is present in the chimeras of formula (lib)
  • its amino end is associated (with an amide linkage) to the carboxyl end of the acidic amino acid tail and its carboxyl end is associated (with an amide linkage) to the amino end of the first polypeptide moiety.
  • the acidic amino acid tail is directly associated with the first polypeptide moiety.
  • the second chimeric polypeptide is of formula (lla) or (lib):
  • SPM is a second polypeptide moiety
  • SAAL is an optional second amino acid linker
  • BAT is a basic amino acid tail having at least one acid amino acid residue having an R-group comprising a carboxyl group.
  • the BAT has a pi between about 9 and 11.
  • the - is an amine bond. It is understood that the SPM can differ from the FPM and that the SPM can be capable of forming an heterodimer or an heterotrimer with the FPM. In some embodiments, the FPM and the SPM can be identical or substantially similar so as to form a homodimer or a homotrimer for example.
  • the second chimeric polypeptide is of formula (Via) or (Vlb):
  • NH 2 - BAT - SPM - COOH (Via) wherein SPM is a second polypeptide moiety; BAT is a basic amino acid tail having at least one acid amino acid residue having an R-group comprising a carboxyl group.
  • the BAT has an isoelectric point (pi) between about 9 and 11.
  • the - is an amine bond.
  • the SPM includes an ectodomain (and in some additional embodiments, a complete ectodomain) of a surface polypeptide.
  • an ectodomain is the domain of a membrane protein that extends in the extracellular space.
  • the ectodomain is involved in binding a ligand and can lead to signal transduction.
  • a “basic amino acid tail” refers to an amino acid tail having one or more basic amino acid residues, such that the pi of the basic amino acid tail is greater than 7.
  • Examples of basic amino acid residues include: arginine, histidine, and lysine.
  • the pi of the basic amino acid tail is less between about 9 and 11.
  • the pi of the acidic amino acid tail is about 10, more preferably 10.05.
  • the basic amino acid tail has a charge which will allow it to interact non-covalently with the acidic amino acid tail and place the SPM is an active conformation.
  • the basic acid tail (BAT) is at least one amino acid and up to 50 amino acid residues in length.
  • the basic amino acid tail (BAT) is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid residues long.
  • the basic amino acid tail is no more than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41 , 40, 39, 38, 37, 36, 35, 34, 33, 32, 31 , 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues long.
  • the BAT has an amino acid sequence of SEQ ID NO: 9 or variants or fragments thereof.
  • the second amino acid linker (SAAL) is present in the chimeras of formula (lla)
  • its amino end is associated (with an amide linkage) to the carboxyl end of the second polypeptide moiety and its carboxyl end is associated (with an amide linkage) to the amino end of the basic amino acid tail.
  • the second amino acid linker (SAAL) is present in the chimeras of formula (lib)
  • its amino end is associated (with an amide linkage) to the carboxyl end of the basic amino acid tail and its carboxyl end is associated (with an amide linkage) to the amino end of the second polypeptide moiety.
  • the basic amino acid tail is directly associated with the second polypeptide moiety.
  • one or more of the carboxyl group of the acidic amino acid tail (AAT) of the first chimeric polypeptide is covalently associated to a first silane linker (FSL) moiety, which in turn is covalently associated with a first hydroxyl group of the surface.
  • One or more of the carboxyl group of the basic amino acid tail (BAT) of the second chimeric polypeptide is covalently associated to a second silane linker (SSL) moiety, which in turn is covalently associated with a second hydroxyl group of the surface.
  • the acidic amino acid tail (AAT) is non-covalently associated (such as charge-charge interaction) with the basic amino acid tail (BAT).
  • the first polypeptide moiety is non-covalently associated with the second polypeptide moiety. In some embodiments, the first polypeptide moiety is non-covalently associated with the second polypeptide moiety in an active conformation.
  • the first chimeric polypeptide has the FAAL, and/or the second chimeric polypeptide has the SAAL. In some embodiments, the first chimeric polypeptide has the FAAL, and the second chimeric polypeptide has the SAAL. In some embodiments, the first chimeric polypeptide has the FAAL, but the second chimeric polypeptide does not have the SAAL. In some embodiments, the first chimeric polypeptide does not have the FAAL, but the second chimeric polypeptide has the SAAL.
  • the first chimeric polypeptide is of formula (la) and the second chimeric polypeptide is of formula (lla). In some embodiments, the first chimeric polypeptide is of formula (lb) and the second chimeric polypeptide is of formula (lla). In some embodiments, the first chimeric polypeptide is of formula (la) and the second chimeric polypeptide is of formula (lib). In some embodiments, the first chimeric polypeptide is of formula (lb) and the second chimeric polypeptide is of formula (lib).
  • the first chimeric polypeptide is of formula (Va) and the second chimeric polypeptide is of formula (Via). In some embodiments, the first chimeric polypeptide is of formula (Vb) and the second chimeric polypeptide is of formula (Via). In some embodiments, the first chimeric polypeptide is of formula (Va) and the second chimeric polypeptide is of formula (Vlb). In some embodiments, the first chimeric polypeptide is of formula (Vb) and the second chimeric polypeptide is of formula (Vlb).
  • the at least one self-assembled multimer is a surface protein (which can include one or more ectodomains for the FPM and/or the SPM).
  • the at least one self-assembled multimer is an activated surface protein.
  • at least one self-assembled multimer is a platelet surface protein.
  • the at least one self-assembled multimer is an integrin dimer.
  • the first chimeric polypeptide comprises a allb polypeptide, functional variants or fragments thereof; and the second chimeric polypeptide comprises a b3 polypeptide, functional variants or fragments thereof.
  • the FPM has an amino acid sequence of SEQ ID NO: 2 or variants or fragments thereof.
  • the corresponding first chimeric polypeptide can have, for example, the amino acid sequence of SEQ ID NO: 1 or variants thereof or fragments thereof.
  • the FPM has an amino acid sequence of SEQ ID NO; 7 or variants thereof or fragments thereof.
  • the SPM has an amino acid sequence of SEQ ID NO: 2 or variants or fragments thereof. In another embodiment, the SPM has an amino acid sequence of SEQ ID NO: 7 or variants thereof or fragments thereof. In such embodiment, the corresponding second chimeric polypeptide can have, for example, the amino acid sequence of SEQ ID NO: 6 or variants thereof or fragments thereof.
  • a variant comprises at least one amino acid difference when compared to the amino acid sequence of the polypeptide polypeptide and exhibits a biological activity substantially similar to the native polypeptide.
  • the polypeptide “variants” have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the polypeptide described herein.
  • the term “percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. The level of identity can be determined conventionally using known computer programs.
  • the polypeptide “variants” have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the biological activity of the polypeptide described herein.
  • One of the biological activity of the cdlb polypeptide is its ability to non-covalently associated with the b3 polypeptide and bind to its ligand (such as fibrinogen).
  • One of the biological activity of the b3 polypeptide is its ability to non-covalently associated with the cdlb polypeptide and bind to its ligand (such as the fibrinogen).
  • the biological of “variants” of the cdlb or the b3 can be assessed by various means known in the art, including, but not limited to antibody-based techniques (flow cytometry, ELISA assay for example) as well as microscopy techniques.
  • the variant polypeptide described herein may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide for purification of the polypeptide.
  • a “variant” of the polypeptide can be a conservative variant or an allelic variant.
  • a conservative variant refers to alterations in the amino acid sequence that do not adversely affect the biological functions of the enzyme.
  • a substitution, insertion or deletion is said to adversely affect the polypeptide when the altered sequence prevents or disrupts a biological function associated with the enzyme.
  • the overall charge, structure or hydrophobic-hydrophilic properties of the polypeptide can be altered without adversely affecting a biological activity.
  • the amino acid sequence can be altered, for example to render the peptide more hydrophobic or hydrophilic, without adversely affecting the biological activities of the enzyme.
  • the polypeptide can be a fragment of polypeptide or fragment of a variant polypeptide.
  • a polypeptide fragment comprises at least one less amino acid residue when compared to the amino acid sequence of the possesses and still possess a biological activity substantially similar to the native full-length polypeptide or functional variants thereof
  • Polypeptide “fragments” have at least at least 100, 200, 300, 400, 500, 600, 700, 800, 900 or more consecutive amino acids of the polypeptide or the polypeptide variant.
  • the polypeptide “fragments” have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the polypeptide described herein.
  • fragments have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the biological activity of the polypeptides and the functional fragments described herein.
  • fragments of the polypeptides can be employed for producing the corresponding full-length enzyme by peptide synthesis. Therefore, the fragments can be employed as intermediates for producing the full-length polypeptides.
  • the carboxyl groups of the chimeric polypeptides are covalently associated to silane linker moieties, which in turn are covalently associated with hydroxyl groups of the surface.
  • the surface is made of a material such as silica, glass, metal, or plastics.
  • the surface has terminal hydroxyl groups.
  • the surface is chemically treated to add terminal hydroxyl groups. The grafting density of hydroxyl groups on the surface can be adjusted, as known in the art, so as to favor or allow the non-covalent association of the acidic and basic amino acid tails as well as the non-covalent association of the first polypeptide moiety and the second polypeptide moiety.
  • the surface is curved. In one embodiment, the surface is spherical. In one embodiment, the surface is a microsphere. In one embodiment, the microsphere is a silica bead. In one embodiment, the microsphere is a glass bead. In one embodiment, the microsphere is a metal bead. In one embodiment, the microsphere is a plastic bead.
  • the surface has a planar surface. In some embodiment, the surface is flat. In one embodiment, the surface is a film. In other embodiments, the surface is a platform. In additional embodiments, the surface is a flat silica surface, a flat glass surface, a flat plastic surface or a flat metal surface.
  • the hydroxyl groups of the surface is covalently associated with the silicone atom of a silane linker moiety.
  • a silane is an inorganic compound having the chemical formula, SiH 4 .
  • a “silane linker” refers to a compound based on SiH 4 , where one or more of the hydrogens is substituted with a group having one or more terminal and/or non-terminal the amine (-NH 2 -) or thiol (-S-) groups.
  • the terminal and/or non-terminal the amine (-NH 2 -) or thiol (-S-) groups of a silane linker moiety is covalently associated with the carboxyl groups of the acidic or basic tails.
  • the silane linker moiety is an amino silane.
  • the amino silane is an amino alkyl silane.
  • the silane linker moiety is 3- trimethoxysilylpropyl) diethylenetriamine (DETA).
  • the silane linker moiety is an thiol silane.
  • the amino silane is a thiol alkyl silane.
  • the surface is a probe surface such as a ⁇ ll ⁇ 3 coupled bead, film, or platform.
  • the ⁇ ll ⁇ 3 coupled bead, film, or platform has application as a molecular probe to identify integrin binding partners, and active conformation of the ⁇ ll ⁇ 3 heterodimer is maintained to allow for binding with platelets to form platelet aggregates.
  • processes of immobilizing at least one self- assembled multimer to a surface is provided.
  • the surface has hydroxyl groups covalently associated with a first and a second silane linker moiety, and the at least one self-assembled multimer is a first and a second chimeric polypeptide.
  • the process includes obtaining a first chimeric polypeptide as described herein, obtaining a second chimeric polypeptide as described herein, and adding the first and second chimeric polypeptide to the surface in a solvent under suitable conditions for the first and second chimeric polypeptides to covalently bond to the surface via a silane linker moiety, wherein the first polypeptide moiety is non-covalently associated with the second polypeptide moiety.
  • the first polypeptide moiety and the second polypeptide moiety form a multimer in an active conformation.
  • the process involves coating the surface with a silane linker by reacting with the hydroxyl groups of the surface.
  • the surface having hydroxyl groups are coated with a silane linker having one or more terminal and/or nonterminal amine (-NH 2 -) or thiol (-S-) groups.
  • the silane linker is DETA and the process involves coating the surface with a DETA linker.
  • the silane linker is not limited to a particular linker and that linkers other than DETA can be used.
  • the process can include pre-treating the surface to provide hydroxyl groups to allow the silane linker to associate with the surface. This can be done, for example, by pre-treating the surface with piranha (70%H 2 S0 4 +30% of 30% H 2 0 2 ), 20 - 40 % NaOH/KOH, or another strong acid or base treatment.
  • pre-treating the surface with piranha (70%H 2 S0 4 +30% of 30% H 2 0 2 ), 20 - 40 % NaOH/KOH, or another strong acid or base treatment.
  • piranha 70%H 2 S0 4 +30% of 30% H 2 0 2
  • 20 - 40 % NaOH/KOH or another strong acid or base treatment.
  • any pre-treatment exposing hydroxyl groups on the surface can be used in the context of the present disclosure.
  • the process involves the recombinant expression of the first and/or the second chimeric polypeptide in a recombinant host cell to obtain the first and/or the second chimeric polypeptide.
  • the process can also include a step of purifying, at least partially, the first and/or second chimeric polypeptide from the recombinant host.
  • the process involves recombinant expression of a surface protein, which can include its ectodomain.
  • the process involves the recombinant expression of an integrin dimer.
  • the process involves activating the multimer form between the first and second chimeric polypeptide.
  • the process can include incubating the surface having the first and second chimeric polypeptides bonded thereon in an activation buffer (which can, in some embodiments, comprise cations).
  • the cations are divalent cations.
  • the cations are magnesium cations (Mg 2+ ).
  • the cations are calcium cations (Ca 2+ ).
  • the cations are manganese cations (Mn 2+ ).
  • rinsing of ferromagnetic silica beads consists of magnetically pelleting the beads, removing the supernatant and resuspending in new solution. (-OH).
  • Activated ferromagnetic silica beads were purchased from Magna MedicsTM and used as received. The beads were first rinsed (3 x) in spectral grade methanol, sonicated for 5 min then rinsed one last time with spectral grade methanol. The rinsing procedure was then repeated with toluene.
  • the beads were dried for 2 h at 180°C then placed in an 80% humidity chamber overnight.
  • a 1% (v/v) solution of neat DETA diluted in anhydrous toluene was prepared in an OTS silanized glass vial.
  • OTS silanized 20 mL scintillation vial silica beads were then immersed in this solution, to a final volume equal to that which was originally aliquoted from the activated bead stock solution, capped and incubated at room temperature on a bench top oscillator overnight.
  • the freshly silanized beads were then rinsed (3 x) with anhydrous toluene, sonicated for 5 min and rinsed again.
  • Activation of ⁇ IIb ⁇ 3 SAMs was achieved by 72 h incubation in activation buffer (1 mM each of CaCI 2 , MnCI 2 and MgCI 2 taken up in PBS).
  • X-ray Photoelectron Spectroscopy Angle-resolved X-ray photoelectron spectroscopy (XPS) to evaluate substrate silanization (SAM formation) and subsequent ⁇ ll ⁇ 3 immobilization was performed with a Theta probe XPS Instrument (ThermoFisher Scientific) located at Surface Interface Ontario (University of Toronto, Toronto, Ontario, Canada). Quartz surfaces were analyzed with monochromated Al Ka X-rays at takeoff angles of 27.5, 42.5, 57.5, and 72.5° relative to the normal. The binding energy scale was calibrated to the C1s signal at 285 eV. Peak fitting and data analysis were performed with the Avantage Data System software package (ThermoFisher ScientificTM) provided with the instrument. Complete XPS data are tabulated Table 1.
  • ⁇ IIb ⁇ 3 Coated Beads Ferromagnetic silica beads were prepared upon formation of DETA adlayers on cleaned silica beads followed by covalent immobilization of ⁇ ll ⁇ 3 ( Figure 1C). Each step of surface preparation was characterized using X-ray photoelectron spectroscopy (XPS) by following the evolution of the characteristic elements of silica (Si and O), DETA and ⁇ ll ⁇ 3 (C and N), (see Table 1). Beside the small signal attributed to unavoidable adventitious carbon and nitrogen contamination, bare silica showed signals for oxygen and silicon at an approximate 2:1 ratio, as would be expected for quartz (Si0 2 ).
  • XPS X-ray photoelectron spectroscopy
  • Ferromagnetic silica particles at each stage of the coating fabrication process were analyzed with flow cytometry for binding to ( Figures 2A and 2B) PSI E1, and ( Figure 2C to 2F) fibrinogen.
  • the integrin is indeed present on the bead surface, and integrin function (ligand binding) is highly conformation dependent, and the integrin must be induced into the high affinity upright conformation prior to ligand binding.
  • ⁇ IIb ⁇ 3 was activated by incubation with a mixture of divalent cations (Mg 2+ ,Ca 2+ and Mn 2+ ) in PBS buffer.
  • the beads were analyzed with flow cytometry for binding of Alexa488TM labelled fibrinogen (native ⁇ ll ⁇ 3 ligand) and PAC-1 (an antibody specific for the active upright conformation of ⁇ ll ⁇ 3) (See Table 2, Figures 2G to 2I).
  • Fibrinogen dose- response curves were also investigated by varying the concentrations of Alexa488TM labelled fibrinogen for binding to activated ⁇ ll ⁇ 3 and DETA (control) particles ( Figure 2E).
  • the DETA signal was subtracted from the activated ⁇ ll ⁇ 3 signal.
  • the activated ⁇ ll ⁇ 3 dose response curve was fitted to a one site specific binding model producing a K d of 78 ⁇ 7 nM.
  • Flow cytometry determined mean fluorescent intensities (MFI) of bare, DETA and activated/inactive ⁇ ll ⁇ 3 beads bound (or nor) with alexa488-fibrinogen (endogenous ligand), FITC-PSI E1 (conformation independent anti— ⁇ ll ⁇ 3 antibody), FITC-PAC 1 (activated conformation specific anti- ⁇ ll ⁇ 3 antibody), FITC-antiCD62 and FITC-anti GPIbp (Isotype controls of the various antibodies), n ⁇ 3
  • MFI mean fluorescent intensities
  • DETA or activated ⁇ ll ⁇ 3 beads were co aggregated with wild type (WT) or Fibrinogen/VWF double deficient (Fg/VWF -/-) platelets by incubating 1x10 8 /ml_ beads with 1x10 8 /ml_ gel filtered platelets in PIPES buffer (25 mM 1 ,4- Piperazinediethanesulfonic acid (PIPES), 140 mM NaCI, 4 mM KCI, 5.5 mM D-glucose pH 7.0). The platelets were then activated by the addition of thrombin to a final amount of 5 units/mL, followed by incubation at RT for 45 min on a rotisserie shaker. The beads were then pulled down, washed and imaged under a Zeiss Axiovert 200 inverted fluorescence microscope at 40x magnification.
  • PIPES buffer 25 mM 1 ,4- Piperazinediethanesulfonic acid (PIPES),
  • Alexa-488 labelled DETA and ⁇ ll ⁇ 3 beads were then mixed in a 1 :1 ratio with gel filtered wild type (WT) murine platelets and activation was initiated with thrombin, the beads were magnetically pulled down and imaged on a fluorescent microscope (Figure 4A). WT platelet aggregates formed under the same conditions in the absence of silica beads are depicted in Figure 4B. The resultant microscopy images clearly show that the ⁇ ll ⁇ 3-coupled beads were incorporated into the platelet aggregates while the DETA beads did not interact with the murine platelets.
  • WT gel filtered wild type
  • Fibrinogen ELISA Assay To ensure similar immobilization levels of recombinant human ectodomain ⁇ ll ⁇ 3 between the ELISA plate and integrin coupled magnetic beads, the wells of a 96-well micro plate (Nunc MaxiSorp) were incubated with the same ⁇ ll ⁇ 3 concentration per surface area as during the preparation of the magnetic beads, 6.6 c 10 4 ⁇ g ⁇ mL _1 cnr 2 .
  • Each well was coated with ⁇ ll ⁇ 3 or control proteins (BSA and b3-/- platelet lysate) by incubation of 6 ⁇ g/ ⁇ L protein in binding buffer (TRIS buffered saline with 0.05 % TWEEN-20 and 1 mM each of MgCI 2 , MnCI 2 and CaCI 2 ) at 4°C overnight. Incubation of 3% skim milk (ED Millipore) and 2% TWEEN-20 for 1 hour at 37°C was used for blocking. See Figures 5A and 5B.
  • binding buffer TriS buffered saline with 0.05 % TWEEN-20 and 1 mM each of MgCI 2 , MnCI 2 and CaCI 2
  • the concentration of ⁇ ll ⁇ 3 per surface area of bead used when coating SAM coated beads is 6.6 x 10 4 ⁇ g ⁇ mL -1 cnr 2 , therefore the wells of 96 well plates used were coated with the same density EDC/NHS quenching achieved by increase pH to 8.6.
  • the same primary antibody was used for both assays, however, the detection antibody for ELISA was HRP labelled and for flow cytometry was FITC labelled.
  • the fibrinogen does response curves are depicted in the inserts of each of Figures 5A and 5B.
  • the flow cytometry-based assay produced a Kd aparant of 0.21 ⁇ 0.03 ⁇ M and CLOD of 0.026 ⁇ 0.002 ⁇ M while ELISA produced a Kd of 2.2 ⁇ 0.4 ⁇ M and CLOD of 0.54 ⁇ 0.07 ⁇ M.
  • the flow cytometry based assay produced a significant increase in performance compared to ELISA, even though a blocking step and a signal amplification- based detection strategy were employed in ELISA. It was postulated that the significant increase in performance observed is due to the SAM and the immobilization strategy which promotes the high-affinity ligand binding conformation of the integrin.

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Abstract

L'invention concerne des surfaces et des procédés pour l'immobilisation de polypeptides multimères qui permet de conserver la fonctionnalité et la conformation active des polypeptides multimères natifs. Le polypeptide multimère est un multimère auto-assemblé et comprend aussi bien un premier d'un second polypeptide chimérique.
PCT/CA2021/050573 2020-04-27 2021-04-27 Multimères de protéines auto-assemblés immobilisés WO2021217251A1 (fr)

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Citations (2)

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US7368295B2 (en) * 2001-08-31 2008-05-06 Fraunhofer-Gesellschaft Zur Foderung Der Angewandten Forschung E.V. Nanoparticles comprising biologically active TNF which is immobilized on the same
EP2903740A2 (fr) * 2012-10-08 2015-08-12 Albert-Ludwigs-Universität Freiburg Procédure d'immobilisation biomoléculaire en une étape et produits en découlant

Patent Citations (2)

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US7368295B2 (en) * 2001-08-31 2008-05-06 Fraunhofer-Gesellschaft Zur Foderung Der Angewandten Forschung E.V. Nanoparticles comprising biologically active TNF which is immobilized on the same
EP2903740A2 (fr) * 2012-10-08 2015-08-12 Albert-Ludwigs-Universität Freiburg Procédure d'immobilisation biomoléculaire en une étape et produits en découlant

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LEE ET AL.: "Facile and high-efficient immobilization of histidine-tagged multimeric protein G on magnetic nanoparticles", NANOSCALE RES LETT, vol. 9, no. 1, 10 December 2014 (2014-12-10), pages 664, XP055867250, ISSN: 1931-7573 *
LIAO ET AL.: "Immobilization of yeast alcohol dehydrogenase on magnetic nanoparticles for improving its stability", BIOTECHNOLOGY LETTERS, vol. 23, 2001, pages 1723 - 1727, XP055867257 *
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